Engineered biocatalysts useful for carbapenem synthesis

Abstract

The present disclosure provides engineered pNB esterase polypeptides useful for the synthesis of the carbapenem antibiotic, imipenem. The disclosure also provides polynucleotides encoding the engineered pNB esterases, host cells capable of expressing the engineered pNB esterases, and methods of using the engineered pNB esterases in the production of imipenem.

What is claimed is: 
     
       1. An engineered polynucleotide encoding an engineered pNB esterase polypeptide having pNB esterase activity, wherein said pNB esterase polypeptide comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 2 and one or more residue differences as compared to SEQ ID NO:2 at residue positions selected from: 108, 115, 116, 130, 193, 214, 219, 273, 276, 321, and 362, wherein the differences at positions 115 and 273 are 115Q/W and 273A/E/T/V, respectively, and optionally wherein said engineered polypeptide further comprises a different residue at position 362. 
     
     
       2. The engineered polynucleotide of  claim 1 , wherein the amino acid residue differences as compared to SEQ ID NO: 2 of the encoded engineered polypeptide at the residue positions 108, 116, 130, 193, 214, 219, 276, 321, and 362 are selected from 108L/Y, 116S, 130T, 164T, 193A/D/E/V, 214G, 219A/D/L/V, 276A/T/L, 321A, and 362A/D/Q/S/V. 
     
     
       3. The engineered polynucleotide of  claim 1 , wherein the amino acid sequence of said encoded polypeptide comprises one or more residue differences as compared to SEQ ID NO: 2 selected from: 108L/Y, 193A/D/E/V, 219A/D/L/V, 273A/E/T/V, and 362A/D/Q/S/V. 
     
     
       4. The engineered polynucleotide of  claim 1 , wherein the amino acid sequence of said encoded polypeptide comprises a residue difference as compared to SEQ ID NO: 2 at position 193 selected from: 193A/D/E/V. 
     
     
       5. The engineered polynucleotide of  claim 4 , wherein the amino acid sequence of said encoded polypeptide comprises a residue difference as compared to SEQ ID NO: 2 at position 193, and wherein said residue is 193V. 
     
     
       6. The engineered polynucleotide of  claim 5 , wherein the amino acid sequence of said encoded polypeptide further comprises residue differences as compared to SEQ ID NO: 2 at positions 219 and 273 selected from 219L/V and 273A/V. 
     
     
       7. The engineered polynucleotide of  claim 6 , wherein the amino acid sequence of said encoded polypeptide further comprises residue differences as compared to SEQ ID NO: 2 at positions 108 and 362 selected from 108L/Y and 362A/D/Q/S/V. 
     
     
       8. The engineered polynucleotide of  claim 2 , wherein the amino acid sequence of said encoded polypeptide comprises a combination of residue differences as compared to SEQ ID NO: 2 selected from:
 193V, 219V, and 273A; 
 108Y, 193D, 219V, 273A, and 362S; 
 108Y, 193V, 219V, 273A, and 362Q; 
 108Y, 115Q, 193V, 219L, 273A, and 362Q; and 
 108Y, 115Q, 193V, 219V, 273A, and 362Q. 
 
     
     
       9. The engineered polynucleotide of  claim 1 , wherein the amino acid sequence of said encoded polypeptide further comprises one or more residue differences as compared to SEQ ID NO: 2 selected from: 116S, 130T, 164T, 214G, 276A/T/L, and 321A. 
     
     
       10. The engineered polynucleotide of  claim 1 , wherein the amino acid sequence of said encoded polypeptide further comprises a residue difference as compared to SEQ ID NO: 2 selected from: 49G, 94G, 96S, 227T, 251V, 267R, 271L, 274L, 313F, 322C/Y, 343V, 356R, 359A, 398L, 412E, 437T, 464A, and 481R. 
     
     
       11. The engineered polynucleotide of  claim 1 , wherein the amino acid sequence of said encoded polypeptide does not comprise a residue difference as compared to SEQ ID NO: 2 at positions 60, 144, 317, 322, 334, 358, and 370. 
     
     
       12. The engineered polynucleotide of  claim 1 , wherein the encoded polypeptide having pNB esterase activity has at least 1.2 fold, 2 fold, 5 fold, 10 fold, 20 fold, 25 fold, 50 fold, 75 fold, 100 fold, or greater increased activity as compared to the polypeptide of SEQ ID NO: 4 in converting compound (2) to compound (1) under suitable reaction conditions. 
     
     
       13. The engineered polynucleotide of  claim 1 , wherein the amino acid sequence of said encoded polypeptide comprises a sequence having at least 90% identity to a polynucleotide sequence selected from SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, and 120. 
     
     
       14. The engineered polynucleotide of  claim 1  comprising a nucleotide sequence having at least 90% sequence identity to a polynucleotide sequence selected from SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, and 119. 
     
     
       15. An expression vector comprising the engineered polynucleotide of  claim 1 . 
     
     
       16. An expression vector comprising the engineered polynucleotide of  claim 14 . 
     
     
       17. The expression vector of  claim 15 , further comprising at least one control sequence. 
     
     
       18. The expression vector of  claim 16 , further comprising at least one control sequence. 
     
     
       19. A host cell comprising the expression vector of  claim 15 . 
     
     
       20. A host cell comprising the expression vector of  claim 16 .